Worldwide, several carcinomas (cancers) stand out as leading killers, with millions of people dying from cancer every year.
This disease is characterized by the proliferation of abnormal or neoplastic cells derived from a normal tissue, forming a tumor mass, the invasion of adjacent tissues by these abnormal cells, and the generation of malignant cells which eventually spread via the blood or lymphatic system to regional lymph nodes and to distant sites (metastasis). With very few exceptions, metastatic disease from a carcinoma is fatal.
Due to the recurrence rates and side effects of current cancer treatment, there is a great need for new therapeutic agents capable of inhibiting neoplastic cell growth. In this scenario, immunoglobulin (Ig) molecules (both IgM and IgG) have been recently reported as sources of bioactive peptides that may display differential anti-microbial, anti-viral and anti-tumor activities in vitro and in vivo (Polonelli et al. PLoS One. 3(6):e2371, 2008; Magliani et al., Curr Med Chem. 6(18):2305-23, 2009). Such internal distribution of bioactive amino acid sequences parallels that of other peptide sequences expressed in receptors of native immunity (Litman et al., Nat. Rev. Immunol., 5: 866-879, 2005). There is, however, no structural similarity among them. Bioactive peptides with sizes ranging from 10aa to 20aa were obtained from the complementarity determining region (CDR) hypervariable domains, from sequences including framework and CDR amino acids, and even from Ig constant regions. To be tested, synthetic preparations of peptides were used which were generally amidated in the C-terminal amino acid. While bioactivity is generally independent of antibody specificity, CDR3 from VH may share antigen binding properties of the original antibody. This is especially true when the antigen is a peptide sequence and less so when it is a carbohydrate. Heavy chain CDR3 peptides that act as the original antibody are called “microantibodies (=microAbs)”.
The conformation of VH CDR3 (H3) peptides was reviewed by Morea et al. (J. Mol. Biol., 275: 269-294, 1998). Unlike the canonical structures of the other five CDRs (H1, H2, L1, L2, L3), the H3 loops have various sizes flanked by C92 and G104 according to Kabat et al. (Sequences of proteins of immunological interest, 5th edition, Public Health Service, N.I.H., Washington, D.C., 1991) numbering in extended sequences of 10 to 22 amino acids.
An example of a bioactive VH CDR3 identified in a monoclonal antibody (mAb) is described hereinafter. Using anti-B16F10 melanoma mAb A4 (Dobroff et al., Hybrid. Hybridomics 21: 321-331, 2002), all six CDRs were tested in the form of synthetic peptides for cytotoxicity. Linear and cyclic extended H3 peptides bound to tumor cells, competed with mAb A4 for binding and they were cytotoxic in cells expressing protocadherin β-13, but not in HL-60 leukemia cells that do not express it (Dobroff et al., Translat. Onc., 3: 204-217, 2010). The advantage of using microAbs is in their small size making it easy to conduct the structure-function analysis and their accessibility to complex reaction sites.
Internal peptide sequences in immunoglobulins (e.g. monoclonal antibodies) belong to constant and variable regions. The CDRs, or the antigen recognizing sequences, although regarded as hypervariable domains, can be individually shared by different immunoglobulins or antibodies. Such similarity among antibodies can involve all CDRs (one each in a family of Ig molecules) with the exception of VH CDR 3. This CDR tends to be unique for a given antibody in its original C92-G104 sequence.
Mattes et al obtained, in 1987, a monoclonal murine IgG1 named MAb MX35, by immunizing mice with a mixture of four fresh ovarian carcinoma specimens. It showed reactivity with approximately 90% of human ovarian epithelial cancers and a limited number of normal tissues as determined by immunohistochemistry (Mattes et al., Cancer Res. 47: 6741-6750, 1987). MX35 MAb has been shown to also react with lung and renal carcinoma cell lines but not with most other carcinoma cell lines as well as cell lines derived from melanomas, astrocytomas, sarcomas, teratocarcinomas, choriocarcinomas and hematopoietic tumors. It reacted with 5/5 specimens of fresh ovarian carcinoma ascites cells by immunofluorescence, with 3/6 benign cysts and 18/18 solid tissue carcinomas by immunoperoxidase staining of frozen sections (Mattes et al., Cancer Res. 47: 6741-6750, 1987).
Subsequently, the localization and biodistribution of radiolabed murine antibody was studied in patients with ovarian carcinoma in phase I clinical trials. F(ab′) of MAb MX35 has been shown to localize to micrometastatic ovarian carcinoma deposits in the peritoneal cavity (Finstad et al., Clin Cancer Res. 3(8):1433-1442, 1997). MAb MX35 and fragments thereof labeled with alpha-particle-emitting nuclide 211astatine (At-a) showed high efficacy in treating micrometastatic tumor growth in a nude mouse model of human ovarian cancer (Elgqvist et al., J Nucl Med. 46 (11): 1907-15, 2005). Also, the MX35 MAb has shown efficient selective tumor localization in vivo as indicated by PET imaging and histology. It also targeted tumors in patients with ovarian cancer with no severe adverse events reported (Hultbom et al., Cancer Biother Radiopharm 21: 373-381, 2006).
More recently, a humanized version of MX35 (called huMX35 or RebMab200) was obtained and is described in an international patent application filed on Jan. 29, 2009 (PCT/US2009/000576).
Throughout this specification, various scientific publications and patents or published patent applications are referenced. The disclosure of all these publications in their entireties is hereby incorporated by reference into this specification in order to more fully describe the state of the art to which the present subject matter pertains. Citation or identification of any reference in this section or any other part of this application shall not be construed as an admission that such reference is available as prior art herein.